Replication of Cryptococcus neoformans in macrophages is accompanied by phagosomal permeabilization and accumulation of vesicles containing polysaccharide in the cytoplasm

Abstract

Cryptococcus neoformans (CN), an encapsulated, ubiquitous environmental yeast, is pathogenic for humans, primarily those with compromised immune function. CN is believed to be a facultative intracellular pathogen. Time-lapsed video microscopy revealed that yeast began to replicate and divide 2 hours after ingestion by J774.16 macrophage cells, with the average cell hosting 10-40 organisms of varying morphologies before ultimately lysing and releasing organisms, either singly or in clumps. Intracellular growth was accompanied by the accumulation of polysaccharide-filled vesicles in the macrophage. Studies with fluorescently labeled dextran revealed that the phagolysosomal compartment became leaky during the course of intracellular infection. Consistent with this observation, phagosomes containing CN had an increased pH relative to similar phagosomes containing inert magnetic beads, as indicated by a colorimetric change in the pH-sensitive Lysosensor dye. Immunocytochemistry revealed differences in the reactivity of polysaccharide elaborated by CN inside macrophages relative to that expressed in vitro. Taken together these results are suggestive of a novel mechanism of intracellular survival by an encapsulated organism, whereby ingestion is followed by damage to the phagosomal membrane resulting in continuity with the cytoplasm, accumulation of polysaccharide-containing vesicles, and possibly, production of a structurally different polysaccharide.

Figure 1

Intracellular budding of C. neoformans strain 24067 in J774 cell. This figure represents individual frames from a composite movie (Movie 1). Arrows indicate CN budding site.

Figure 2

Intracellular shedding and accumulation of C. neoformans polysaccharide 18 h after phagocytosis. (A) Phase image of infected cell. (B) Corresponding fluorescent image. Immunodetection of CN polysaccharide was with fluorescently labeled primary Ab to GXM. CN was ingested by means of opsonization with human complement. (C) Uninfected control monolayer of J774 cells. (D) Horseradish peroxidase staining of cells infected with CN strain 24067. CN was ingested by means of opsonization with GXM-specific Ab (IgG1) followed by detection with GXM-specific Ab of a different isotype (IgG3). Arrows in B and D point to released GXM. Original magnification is ×90.

Figure 3

Dissolution of J774 phagosomal membrane by intracellular C. neoformans opsonized with complement (20 K magnification). Left arrowhead indicates an area of in tact phagosomal membrane. Right arrowhead points to dissolved phagosomal membrane. Arrow points to nuclear membrane that is intact. P, polysaccharide; N, host cell nucleus.

Figure 4

Phagosomes containing C. neoformans become permeable to macromolecules. (A) Fluorescence image of J774 cells that have taken up fluorescent dextran by means of pinocytosis. Arrow points to dextran leaking from CN-containing phagosome. (B) Phase image of A. (C) Fluorescence image of infected J774 cell microinjected with fluorescent dextran. (D) Phase image of C. The diameter of two CN-containing phagosomes has been traced with bars and layered onto the fluorescence image of the same plane of focus for comparison. (E) Stacked z-series of confocal fluorescence images of inert beads within intact phagosome. (F) Similar confocal series of CN-containing phagosome where tongue-like projections of leaking polysaccharide are indicated by arrows. (Movies 2 and 3 correspond to E and F, respectively.)

Figure 5

C. neoformans phagosome becomes less acidic with time as determined by changes in pH indicator color. J774 cells with ingested magnetic beads at 30 min and 3 h (A and C, respectively); corresponding phase images (B and D). Arrows in A and C point to beads. J774 cells with intracellular CN at 30 min and 3 h (E and G, respectively); corresponding phase images (F and H). Arrows in E and G point to CN. Yellow indicates acidic pH.

Figure 6

Intracellular growth of CN strain 24067 in mouse primary alveolar macrophage leads to host cell lysis. A–D represent single frames from Movie 4. Arrows indicate morphological switch to oval budding pattern (A–C) and rupture of host cell in D (Movie 4).

Figure 7

Proposed model of events leading to C. neoformans intracellular survival within macrophages. After phagocytic uptake (1), phagolysosomal fusion occurs (2). Late-stage trafficking markers, such as LAMP-1, are represented in green (3). CN appears to dissolve the phagosomal compartment and intracellular replication ensues (4), while CN-derived polysaccharide builds up within the host cell (5). Ultimately, the host cell ruptures, releasing the newly replicated CN (6).